Not applicable.
Cholesterol and other lipids are transported in body fluids by low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Substances that effectuate mechanisms for lowering LDL-cholesterol may serve as effective antihypercholesterolemic agents because LDL levels are positively correlated with the risk of coronary artery disease.
MEVACOR (lovastatin; mevinolin) and ZOCOR (simvastatin) are members of a group of active antihypercholesterolemic agents that function by inhibiting the rate-limiting step in cellular cholesterol biosynthesis, namely the conversion of hydroxymethylglutarylcoenzyme A (HMG-CoA) into mevalonate by HMG-CoA reductase.
The general biosynthetic pathway of a naturally occurring HMG-CoA reductase inhibitor has been outlined by Moore, et al., who showed that the biosynthesis of mevinolin (lovastatin) by Aspergillus terreus ATCC 20542 begins with acetate and proceeds via a polyketide pathway (R. N. Moore, et al., J. Amer. Chem. Soc. 107:3694-3701, 1985). Endo, et al. described similar biosynthetic pathways in Pencillium citrinum NRRL 8082 and Monascus ruber M-4681 (A. Y. Endo, et al., J. Antibiot. 38:444-448, 1985).
The recent commercial introduction of microbial HMG-CoA reductase inhibitors has fostered a need for high yielding production processes. Methods of improving process yield have included scaling up the process, improving the culture medium and simplifying the isolation.
Previous attempts to increase the biosynthesis of HMG-CoA reductase inhibitors at the level of gene expression have focused on increasing the concentration triol polyketide synthase (TPKS), a multifunctional protein with at least six activities as evidenced by the product of the enzymatic activity (Moore, supra, 1985). TPKS is believed to be the rate-limiting enzymatic activity(ies) in the biosynthesis of the HMG-CoA reductase inhibitor compounds.
U.S. Pat. No. 5,744,350 identifies a DNA encoding triol polyketide synthase (TPKS) from Aspergillus terreus. xe2x80x9cNPKSxe2x80x9d is now preferred to TPKS as the acronym for xe2x80x9cnonaketide polyketide synthase.xe2x80x9d
In one embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing organism. The method comprises the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment, preferably comprising nucleotides 579-33,000 of SEQ ID NO:18 and 1-5,349 of SEQ ID NO:19. The nucleic acid sequence is transcribed and translated and an increase in lovastatin production occurs. Preferably, this increase is at least 2-fold.
In a preferred form of the present invention, the lovastatin-producing organism is selected from the group consisting A. terreus ATCC 20542 and ATCC 20541.
In another embodiment, the method comprises the step of transforming the organism with the corresponding D4B segment isolated from a non-A. terreus lovastatin-producing organism.
In another embodiment, the present invention is a method of increasing the production of lovastatin in a lovastatin-producing organism, comprising the step of transforming the organism with the LovE gene, wherein the nucleic acid sequence is transcribed and translated and wherein an increase in lovastatin production occurs.
In another embodiment of the present invention, one may increase the production of monacolin J in a non-lovastatin-producing organism comprising the steps of transforming the organism with a nucleic acid sequence comprising the D4B segment. As a further step, one may additionally transform the organism with an entire LovF gene. If the entire LovF gene is added to the D4B segment, the organism will produce lovastatin.
In another embodiment, the present invention is the lovastatin production gene cluster, preferably SEQ ID NOs:18 and 19, and the individual genes comprising that cluster.
It is an object of the present invention to provide a method for increasing lovastatin and monacolin J production in both lovastatin-producing and non-lovastatin producing organisms.
Other objects, features and advantages of the present invention will become apparent after review of the specification, claims and drawings.